Embodiments of the present invention are directed to a human pancreatic cell line transfected so as to express xcex2-cell differentiation factor IDX-1. Cells so transfected respond to glucagon-like peptide-1 by differentiating into insulin-secreting xcex2 cells.
Embodiments of the present invention are directed to a human pancreatic cell line transfected so as to express xcex2-cell differentiation factor IDX-1. Cells so transfected respond to glucogon-like/peptide-1 by differentiating into insulin-secreting xcex2cells.
Insulin is essential for proper metabolism in humans: in addition to its familiar role as the chief regulator of blood sugar levels in humans, it is essential for carbohydrate, lipid, and protein metabolism, as well. Pancreatic beta (xcex2) cells of the islets of Langerhans, epithelial cells dispersed throughout the pancreas, secrete insulin. When xcex2 cells are destroyed or their function impaired, insulin production declines, and diabetes results.
The most common form of diabetes, presenting in nearly a million new cases every year in the United States, is type II diabetes. Type II refers to a group of disorders characterized by high blood levels of glucose (hyperglycemia) and a resistance to insulin. Administering insulin to such patients tends not to produce its usual effect: in healthy individuals, insulin increases glucose uptake by skeletal muscle and decreases glucose production in the liver; in individuals with type II diabetes, insulin tends not to do so. Many patients with type II diabetes, therefore, do not respond well to insulin therapy, even when it is administered at high doses.
Drugs that promote insulin secretion or that lower glucose levels by other means are commonly prescribed to treat patients with type II diabetes. Sulfonylureas are the principal drugs prescribed to such patients. They stimulate insulin production by directly stimulating xcex2 cells; the effectiveness of such drugs therefore depends on the number of functioning xcex2 cells remaining in the pancreas. Repaglinide also stimulates insulin production by stimulating xcex2 cells, but differs structurally from the sulfonylureas. Other drugs, such as troglitazone (known better by its brand name, REZULIN(copyright)) and metformin, lower glucose levels by reducing glucose production in the liver and by promoting insulin sensitivity. Another drug, acarbose, inhibits digestive enzyme secretion and thereby delays digestion of carbohydrates (which when broken down in the body ultimately yield glucose). The efficacy of these drugs is tested first in vitro using existing cell lines that seek to model insulin-secreting xcex2 cells. None of these cell lines provides a satisfactory model, however, because they lose their responsiveness to glucose. As a result, in vitro studies of insulin-secreting drugs currently provide only limited information regarding their efficacy.
Understanding the function and development of insulin-secreting xcex2 cells is a critical step in developing better drugs to treatxe2x80x94and ultimately curexe2x80x94diabetes. Pancreatic endocrine and exocrine cells (the cells that secrete insulin and other hormones) originate from a precursor epithelial cell during the development of the pancreas. G. Teitelman and J. K. Lee, xe2x80x9cCell lineage analysis of pancreatic islet cell development: glucagon and insulin cells arise from catecholarninergic precursor present in the pancreatic duct.xe2x80x9d Dev. Biol. 121:454-466, 1987; R. L. Pictet, W. R. Clark, R. H. Williams, and W. J. Rutter, xe2x80x9cAn ultrastructual analysis of the developing embryonic pancreas.xe2x80x9d Dev. Biol. 29:436-467, 1972 (the foregoing publications, and all other publications cited herein, are incorporated by reference in their entirety). Various differentiation factors are required to achieve the mature phenotype characteristic of islet beta xcex2-cells.
New xcex2-cells are formed from existing islets and from ductal epithelial cells. The latter source has greater intrinsic biological relevance. Indeed, the possibility of differentiating insulin-secreting cells from non-endocrine cells supports the hypothesis that the biological source (pancreatic ductal epithelium) for this compensatory mechanism may be present even in the setting of a generalized destruction of the entire population of islet xcex2-cells. This is strongly supported by recent studies demonstrating that primary cultures of epithelial ductal cells (from human and mouse pancreas) are susceptible to undergo differentiation into endocrine cells. V. K. Ramiya, M. Maraist, K. E. Arfors, D. A. Schatz, A. E. Peck, J. G. Conmelius, xe2x80x9cReversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells.xe2x80x9d Nature Medicine, 6(3):278-82, 2000; S. Bonner-Weir, M. Taneja, G. C. Weir, K. Tatarkiewicz, K. H. Song, A. Sharma, J. J. O""Neil, xe2x80x9cIn vitro cultivation of human islets from expanded ductal tissue. Proc. Natl. Aca. Sci. USA, 14:7999-8004, 1997.
Growth and differentiation of islet xcex2-cells is not limited to the embryological state. A constant remodeling of size and function of the islets of Langerhans occurs during the entire life of individuals and is likely to play an essential role in the prevention of diabetes. In adult rats, two independent pathways are utilized for the proliferation of pancreatic endocrine cells: in the first pathway of proliferation, new endocrine cells arise from the division and differentiation of cells within the islets; in the second pathway, the islets cells originate from precursor cells located in the pancreatic ductal epithelium. S. Bonner-Weir, L. A. Baxter, G. T. Schuppin, F. E. Smith, xe2x80x9cA second pathway for regeneration of adult exocrine and endocrine pancreas. A possible recapitulation of embryonic development.xe2x80x9d Diabetes 42:1715-1720, 1993.
It has yet to be determined whether in the normal ductal epithelium there are different populations of cells, some of which are capable of differentiating into endocrine cells, while others have merely a structural role in defining the epithelial wall It is also possible that all pancreatic ductal epithelial cells could represent a not-fully-differentiated population of cells capable of acquiring a new phenotype Under specific stimuli, but this, too, has yet to be determined; at present, this possibility is a matter of speculation. It is likely that a coordinated activation of multiple differentiation factors, in a fashion similar to the sequence of events occurring during fetal development, is required for the cellular growth of the endocrine pancreas of adults. The mechanism (or mechanisms) for the activation of such a complex regulatory network in adulthood is poorly understood.
An incretin hormone, glucagon-like-peptide-1 (GLP-1), is believed to play a role in the development of the pancreas, though researchers have disagreed as to precisely what this role is. A decade ago, for example, U.S. Pat. No. 5,120,712, the entirety of which is incorporated by reference, stated that xe2x80x9cThe failure to identify any physiological role for GLP-1 caused some investigators to question whether GLP-1 was in fact a hormone and whether the relatedness between glucagon and GLP-1 might be artifactual.xe2x80x9d Researchers have more recently learned that GLP-1 has a function in rats. Bonner-Weir et al., for example, demonstrated that an analog of the incretin hormone glucagon-like-peptide-1 (GLP-1), termed exendin4, was able to increase islet mass in adult animals previously subjected to subtotal pancreatectomy. G. Xu, D. A. Stoffers, J. F. Habener, S. Bonner-Weir, xe2x80x9cExendin-4 stimulates both beta-cell replication and neogenesis, resulting in increased beta-cell mass and improved glucose tolerance in diabetic rats.xe2x80x9d Diabetes 48:2270-2276, 1999. Similarly, the inventor has demonstrated that treating glucose-intolerant aging Wistar rats with GLP-1 restored normal glucose tolerance and induced islet cell proliferation. Y. Wang, R. Perfetti, N. H. Greig, H. W. Holloway, K. A. DeOre, C. Montrose-Rafizadeh, D. Elahi, J. M. Egan, xe2x80x9cGlucagon-like peptide-1 can reverse the age-related decline in glucose tolerance in rats.xe2x80x9d J Clin Invest 99:2883-2889, 1997.
Islet duodenal homeobox-1 (xe2x80x9cIDX-1,xe2x80x9d also known variously as IPF-1/STF-1 and PDX-1) is a homeodomain protein and an insulin gene transcription factor expressed in the early pancreatic gland of the embryo. During pancreatic islet development, IDX-1 plays an important role in determining islet cell differentiation. It is the early IDX-1 gene expression during embryogenesis, coupled with the activation of other transcription factors (for example, NeuroDBeta 2, Pax 4, etc.), that determine the pancreatic endocrine hormone production. In adult (mature) animals, the expression of IDX-1 is repressed in the majority of pancreatic cells, with the exception of the xcex2- and xcex4-cells (somatostatin-secreting cells) of the islets of Langerhans.
IDX-1 plays an important role in the development and functioning of the pancreas, though researchers have yet to elucidate precisely what that role is. Mice lacking islet IDX-1, for example, fail to develop a pancreas. J. Jonsson, L. Carlsson, T. Edlund, and H. Edlund, xe2x80x9cInsulin-promoter factor 1 is required for pancreas development in mice. Nature 371:606-609, 1994. Islet-1, a LIM homeodomain-containing protein, is necessary for the development of the dorsal pancreas and is required for the generation of islet cells. U. Ahigren, S. L. Pfaff, T. M. Jessell, T. Edlund, and H. Edlund, xe2x80x9cIndependent requirement for ISL1 in formation of pancreatic mesenchyme and islet cells.xe2x80x9d Nature 385:257-260, 1997. Inactivation of BETA2/NeuroD or Pax4 genes cause a striking reduction in the number of insulin-producing cells and a failure to develop mature islets. F. J. Naya, H. P. Huang, Y. Qiu, H. Mutoh, F. J. DeMayo, A. B. Leiter, M. J. Tsai: xe2x80x9cDiabetes, defective pancreatic morphogenesis, and abnormal enteroendocrine differentiation in BETA2/NeuroD-deficient mice.xe2x80x9d Genes. Dev. 11:2323-2334, 1997.
The mechanisms regulating proliferation and differentiation of the pancreatic hormone-producing cells and the chronology of these biological events are still largely undetermined. The sequence of events described herein suggests that the ability of regulating glucose uptake by the islet-specific glucose transporter GLUT2 is the first step necessary for the xe2x80x9csensitizationxe2x80x9d of the regulatory region(s) of the insulin gene to glucose. This would then promote the transcription of insulin mRNA. GLP-1-dependent activation of IDX-1 would further commit these cells to a xcex2-cell-like pathway of differentiation by inducing the synthesis of glucokinase, the chief element of the glucose-sensing machinery of the islets of Langerhans.
Researchers have learned much of the role of GLP-1 and IDX-1 in the rat and mouse, where knock-out mouse or other animal models are available to study the role of these hormones. Researchers know little of the role GLP-1 and IDX-1 in the development of human insulin-secreting cells, or of their interaction with other hormones present in the endocrine system. There is therefore an important need in the art for an analytical tool that permits researchers to elucidate the role of GLP-1 and IDX-1 in humans. A human model would be of immense importance in testing theories of endocrine development, in evaluating antidiabetic drugs, and developing new approaches to treat diabetes.
It is an object of the invention to provide a model and analytical tool to study human insulin-secreting cells. It is a further object of the invention to provide an analytical tool that elucidates the role of GLP-1 and IDX-1xe2x80x94two hormones whose roles are incompletely understood in the development and functioning of human insulin secreting cells. It is a still further object of the invention to provide a human model that permits one to test theories of endocrine development, to evaluate antidiabetic drugs, and to develop new approaches to treat diabetes.
Disclosed herein is a novel insulin-secreting human cell line. The cell line of the invention is based on the surprising discovery that human pancreatic cells, when transfected with IDX-1 and cultured in GLP-1, differentiate into insulin-secreting xcex2 cells. These cells moreover behave in many respects as human xcex2 cells do in vivo. In a significant respect, these cells secrete insulin in a dose-dependent manner; that is, the more glucose these cells are exposed to, the more insulin they secrete. This important feature regarding this cell line makes it an ideal model to test antidiabetic drugs, such as sulfonylureas, repaglinide, and other drugs, that are commonly administered to patients with type II diabetes.
To date, no one has succeeded in constructing a cell line comprising human cells that secrete insulin in a dose-dependent manner. Such a cell line, disclosed for the first time herein, provides an important tool for investigating the function of the endocrine system, its development, and drugs that can affect it.